Unknown,Transcriptomics,Genomics,Proteomics

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RNA-Seq of ERα-positive breast cancer cell lines to measure the interplay of fulvestrant treatment and hypoxic conditions

ABSTRACT: Estrogen receptor alpha (ERα) and hypoxia-inducible factors (HIFs) are key regulators of transcriptional programmes in breast cancer. To investigate their interplay, we performed RNA sequencing on ERα-positive breast cancer cell lines (MCF-7 and T-47D) following treatment with the selective estrogen receptor degrader fulvestrant and/or exposure to hypoxic conditions (1% O₂). Cells were cultured for 96 hours in the presence or absence of 100 nM fulvestrant, with the final 48 hours spent under normoxia or hypoxia. RNA was extracted from biological triplicates and sequenced using Illumina short-read technology (≥20 million reads/sample). Differential gene expression analysis revealed distinct transcriptional programmes regulated by ERα and hypoxia, as well as a large set of overlapping genes modulated by both conditions. Fulvestrant-induced ERα degradation suppressed canonical estrogen-responsive genes (e.g. GREB1, TFF1) and upregulated EPAS1 (HIF-2α). Hypoxia induced well-characterised HIF targets (e.g. CA9, VEGFA).

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER: Andrew Holding

PROVIDER: E-MTAB-15331 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Approximately 30% of metastatic breast cancers harbor estrogen receptor α (ERα/ESR1) mutations that are associated with resistance to endocrine therapy and shorter patient survival. How these ERα mutations elicit an aggressive tumor phenotype was largely unknown. Recently, we reported cell lines in which the wild-type ESR1 gene is replaced by the two most common ERα mutations, ERαY537S and ERαD538G. Here we use transcriptome analysis to demonstrate that ERαY537S cells exhibit a unique expression profile and phenotype, and identify an important role for Myc in the increased growth rate, ligand independent cell proliferation, and partial resistance to tamoxifen and fulvestrant of cells expressing the ERαY537S and ERαD538G mutations. Notably, the T47DERαY537S (TYS) cells exhibit a markedly different phenotype and transcriptome than the parental T47D cells and T47DERαD538G (TDG) cells. Consistent with their estrogen-independent proliferation, RNAseq shows the TYS and TDG cells exhibit estrogen independent gene expression patterns. Gene set enrichment analysis showed Myc target pathways are highly induced in both mutant cell lines. Unlike the parental T47D cells, in TYS and TDG cells Myc mRNA and protein exhibit constitutive, fulevestrant-resistant expression. Moreover, chromatin immunoprecipitation (ChIP) showed that ERαY537S and ERαD538G exhibit constitutive, fulvestrant-resistant, recruitment to the Myc enhancer region. Myc knockdown showed that Myc protein is required for ligand independent proliferation of TYS and TDG cells. Constitutive MYC expression in T47D cells, to levels similar to those in TYS and TDG cells, partially recapitulated the aggressive phenotypes of the TYS and TDG cells. Our study demonstrates a previously undescribed role for constitutive and antiestrogen resistant Myc expression in the aggressive phenotype exhibited by breast cancer cells expressing ERα mutations and provides powerful tools for identifying pathways that contribute to the ERα mutant phenotype.

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RNA-Seq of ERα-positive breast cancer cell lines to measure the interplay of fulvestrant treatment and hypoxic conditions

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